The project is presently concentrating on quantitative analysis of pneumococcal adherence and phagocytosis by human peripheral blood monocytes. A new method was developed that employs two fluorescent labels and dual laser flow cytometry that distinguishes between attached and ingested bacteria. The pneumococcal are covalently labeled with Lucifer Yellow which fluoresces green independent of pH. Surface bound pneumococci are labeled with biotinylated F(ab')2 anti-capsular or anti-Lucifer antibodies to study opsonization requirements for adherence and rate of ingestion. We have also found that CR1 mediates binding and ingestion of serum opsonized pneumococci.